Countermeasure protocols of mustard decontamination and neutralization may protect exposed surfaces of the skin, eyes, and lungs when administered immediately after exposure. However, this may not be adequate to address the delayed onset of myelosuppression and disrupted hematopoiesis associated with mustard exposure. The impact of the current proposal is to provide a comprehensive countermeasure regimen that addresses both the acute and delayed detrimental effects of mustard exposure mediated by acute skin inflammation and the innate immune system, specifically macrophage hyper-activation. NM-mediated tissue injury of the skin results in rapid epidermal disruption and vascular leakage resulting edema and painful inflammation. Following the initial injury is an influx of activated inflammatory monocytes and macrophages. Release of macrophage-mediated soluble signaling molecules promotes a pro- inflammatory state which amplifies other cytotoxic activities including release of reactive nitrogen species iNOS, reactive oxygen species, and induction of proteolytic enzymes. Furthermore, these activated iNOS- producing macrophages are critically involved in mediating and exacerbating systemic toxicities. We propose to use a multidisciplinary translational approach to modulate skin inflammation and macrophage activity using in vivo techniques in mouse and human studies. We will study the effect of spironolactone and bisphosphonates (BPs) in combination with vitamin D to inhibit the production of pro- inflammatory factors including; TNF?, and iNOS in the skin and in activated macrophages. These drugs have been in clinical use for many years with known safety and toxicity profiles. Spironolactone is a potassium- sparing diuretic (water pill) that has been safely used for five decades to treat essential hypertension, fluid overload and edema in patients with congestive heart failure and other kidney pathologies. While much information has been accumulated on the physiopathology of spironolactone on the mineralocorticoid receptor (MR) and the adrenal glands, the effect of MR ligands on non-classical targets such as the skin has found new and exciting potential applications. We hereby propose that spironolactone being a weak diuretic has sufficient capacity to reduce water content of edematous skin lesions as a way to accelerate wound healing. The use of spironolactone may have benefits in limiting edema and painful skin inflammation. BPs are medications that has been used for over 40 years. In addition to the skeletal bones, these drugs deposit in the skin, liver, spleen, and bone marrow which aligns with our research interest as they represent organs and tissues that may be reservoirs and/or targets for activated macrophages. Furthermore, BPs deposit and have long-lasting effects in the tissue. We propose to use BPs in conjunction with high dose vitamin D3 as countermeasures to NM exposure. Lastly, as a systemic treatment for the most toxic exposure, we propose to use a novel immune modifying microparticle (IMPs) which are derived from clinical polymers. Upon binding and engulfment of IMPs, macrophages are sequestered for degradation in the liver and spleen. The strategy with IMPs represents a new aspect of this grant to target severe systemic toxicity. To validate and determine the translational significance of BPs and vitamin D3 in suppressing skin inflammation following mustard exposure, we designed a human clinical study using UV (solar spectrum radiation) as a surrogate for mustard exposure in healthy human subjects to expedite future translational studies. It is well known that sunburn exposure leads to redness, swelling, vesication, and activation of macrophages in the skin including upregulation of iNOS and TNF?. Information from this human clinical study will be informative and confirmatory of the use of our proposed countermeasures. Our assembled team involve co-investigators from different centers within and outside the university and hospital. The team of co-investigators and consultants will have oversight of the project, helping to identify new opportunities and complementary approaches drawing from expertise in: dermatopharmacology; inflammation and macrophages; vitamin D; skin and tissue pathology; clinical trials implementation and regulatory compliance; statistics and epidemiology - all critical to the translation of this study into a practical intervention. This is a novel and highly translational approach to develop potential countermeasure to address key morbidity and mortality factors from mustard exposure. Spironolactone, bisphosphonates, and vitamin D are a widely available and inexpensive and, if shown to be beneficial, could have a broad public health impact in the event of chemical weapon exposure.